JOURNAL OF BACTE1RIOLOGY, Sept., 1965 Vol. 90, No. 3 Copyright @ 1965 American Society for Microbiology Printed in U.S.A, Physiological Effects of a Constitutive Tryptophanase in Bacillus alvei J. A. HOCH AND R. D. DEMOSS Department of Microbiology, University of Illinois, Urbana, Illinois Received for publication 8 April 1965 ABSTRACT HOCH, J. A. (University of Illinois, Urbana), AND R. D. DEMoss. Physiological effects of a constitutive tryptophanase in Bacillus alvei. J. Bacteriol. 90:604-610. 1965. -Tryptophanase synthesis in B. alvei is not under the control of tryptophan and is not subject to catabolite repression. Exogenously supplied tryptophan was converted to indole by tryptophanase, and was excreted into the culture medium. The amount of indole excreted was dependent upon the concentration of tryptophan supplied. At intermediate levels of tryptophan (5 to 15 ,jg/ml), the excreted indole was completely reutilized by the cell, in contrast to the result with higher levels. Indole reutil zation was shown to be dependent upon a functional tryptophan synthetase. In the absience of exogenous tryptophan, indole was excreted into the culture medium at an earlier phys- iological age. The early indole was shown not to be a consequence of tryptophanase action. The early indole accompanied uniformly the normal process of tryptophan biosynthesis, and the fission of indole-3-glycerol phosphate was suggested as the origin of the excreted indole. The enzyme tryptophanase from Escherichia MATERIALS AND METHODS coli has been the subject of considerable research Bacteria. B. alvei ATCC 6348 was obtained from in recent years (Burns and DeMoss, 1962; New- the American Type Culture Collection. A non- ton and Snell, 1964). The enzyme cleaves tryp- mucoid variant which appeared on nutrient agar tophan to indole, pyruvate, and ammonia, with plates was isolated from this strain and used in the pyridoxal phosphate as a cofactor. Tryptophan- experiments reported here. This strain, designated ase in E. coli may serve only in a catabolic role, strain F, has the advantage of being more sus- to metabolizable ceptible to disruption by physical methods than degrading tryptophan pyruvate the parent strain. and excreting indole into the culture medium. Auxotrophic mutants, induced with ultraviolet Newton and Snell (1964) have shown that tryp- light, were selected by the penicillin technique of tophanase may also synthesize tryptophan from Nester, Schafer, and Lederberg (1963). The mu- indole and serine. A mutation to constitutivity tants used in this study are described in Table 1. for tryptophanase in a mutant with the trypto- Accumulated intermediates were identified by phan biosynthetic loci deleted will permit indole paper chromatography. to replace tryptophan as a growth requirement. Culture media. Starter cultures for all experi- Since E. coli is the only organism in which ments were grown on 2% (w/v) Trypticase (BBL), it pH 7.0, supplemented with 10 ,g/ml of thiamine tryptophanase has been studied, was of interest hydrochloride. The mineral salts prepared accord- to determine whether the enzyme was present ing to Smith and Yanofsky (1962) were supple- in other unrelated bacteria. Assuming that pro- mented with 10 ,g/ml of thiamine hydrochloride. duction of indole is a manifestation of tryp- Acid-hydrolysed casein (Nutritional Biochemicals tophanase activity, we have investigated a num- Corp., Cleveland, Ohio), 1% (w/v), was employed as the carbon source. ber of Bacillus species for this characteristic. One Tryptophanase assay. Tryptophanase activity species, B. alvei, produced indole and was found was assayed according to Boezi and DeMoss to exhibit true tryptophanase activity. (1961). The reaction mixture contained: pyri- In the course of investigation of tryptophanase doxal-5-phosphate, 40 pAg; potassium phosphate in B. alvei, its physiological state in the organism buffer (pH 8.0), 300 j,moles; and enzyme solution made to 1.5 ml with distilled water. The reaction was observed to have effects on tryptophan bio- mixture was overlaid with 4 ml of toluene and synthesis; it may fulfill a regulatory role in tryp- shaken at 37 C for 5 min in a 50-ml Erlenmeyer tophan biosynthesis. flask. The reaction was initiated with 0.5 ml of 604 VOL. 90, 1965 CONSTITUTIVE TRYPTOPHANASE IN B. ALVEI 605 0.02 M L-tryptophan and allowed to incubate at TABLE 1. Characteristics of Bacillus alvei mutants* 37 C for 1 hr. The reaction was stopped by adding 0.1 ml of 2 N NaOH and shaking for 5 min. Mutant no. Growth response Accumulation The indole present in the toluene layer was deter- mined according to Yanofsky (1955). Indole for- TS-2 An, In, Try mation in the assay is linear with respect to time TS-15 In, Try IG for at least 5 hr, and is linear with respect to en- TS-19 In,t Try In zyme concentration. One unit of enzyme is defined as that amount of * Abbreviations: An, anthranilic acid; In, enzyme forming 0.1 jAmole of indole per hr. Spe- indole; Try, tryptophan; IG, indole-3-glycerol. cific activity is expressed as units of enzyme per t Leaky on indole. absorbancy unit at 660 m,u of the cell suspension. Determination of indole. To determine indole in TABLE 2. of tryptophan on tryptophanase small samples of the culture, advantage was taken Effect of the high extinction coefficient of the indole-p- synthesis* dimethylaminocinnamaldehyde (PDAC) complex (Scott, 1960). The PDAC reagent consists of 5 Addition Amount Growth 1.activitySpecific parts of a 5% (w/v) solution of PDAC in 95% (v/v) ethyl alcohol and 12 parts of acid-alcohol ,ug/ml (16 ml of concentrated H2SO4 and 200 ml of 95% Tryptophan ............. 0 0.260 3.61 ethyl alcohol). To 0.3 ml of culture, 1.0 ml of 10 0.244 2.79 PDAC reagent was added, and after thorough mix- 50 0.258 3.33 ing the tubes were centrifuged for 5 min at 3,400 X 100 0.250 3.60 g to sediment debris. The clear supernatant fluid 500 0.250 3.28 was removed, and the absorbancy at 625 m, was 1,000 0.201 2.98 read in a Zeiss PMQ II spectrophotometer after Anthranilic acid ......... 200 0.337 3.17 10 min. Indole concentration was estimated from a Indole .................. 50 0.305 3.21 previously constructed standard curve. In some cases the indole was determined after extraction * Cells were harvested after 12 hr, and were into toluene. Either assay gives comparable re- assayed immediately. Growth is presented as sults. absorbancy at 660 m,u; specific activity is ex- Determination of tryptophan. Tryptophan was pressed as units of enzyme per absorbancy unit determined in culture supernatant fluids by the at 660 m,u. method of Frank and DeMoss (1957) with par- tially purified E. coli tryptophanase. Measurement of absorbancy. The absorbancy of (Table 2). Indole and anthranilic acid similarly cultures was measured at 660 m,u, either in a Zeiss have no effect. Such a situation can be envisioned PMQ II spectrophotometer or in an Evelyn photo- if tryptophan is overproduced by the organism, electric colorimeter. thus rendering the exogenous level of tryptophan Measurement of radioactivity. Indole-2-C14 insignificant. On the contrary, tryptophan auxo- (Calbiochem) was converted enzymatically to L-tryptophan-2-C14. In a typical experiment, a trophs grown on a limiting concentration of tryp- sample of culture was sedimented at 8,700 X g for tophan (e.g., 10 ,ug/ml) do not have enzyme levels 15 min. The cells were suspended in 5% (w/v) lower than the wild type. An alternative expla- trichloroacetic acid, and were allowed to stand at nation would suggest that the organism lacks a 4 C for at least 30 min. A sample of this cell sus- tryptophan permease. Although no permease pension was filtered on a membrane filter (no. studies were undertaken, this possibility is not B-6, Carl Schleicher & Schuell Co., Keene, N.H.), likely because copious amounts of indole accu- and the filter was allowed to dry. The supernatant mulate in the presence of exogenous tryptophan. fluid from the initial centrifugation was extracted with toluene, and samples of the toluene layer Thus, tryptophanase synthesis is not under were assayed for indole and radioactivity. A Tri- the control of tryptophan but rather is constitu- carb Liquid Scintillation Spectrometer was em- tive. It might be noted that the specific activity ployed for all radioactivity counting. of fully induced E. coli is approximately 200-fold higher than that of B. alvei. RESULTS Effect of tryptophan on indole excretion. When B. alvei was grown in the presence of Effect of tryptophan on enzyme level. E. coli tryp- tryptophan, tophanase is inducible by high levels of trypto- indole was excreted into the culture medium and phan; in its absence, only a minute amount of presumably arose from tryptophanase action. enzyme is synthesized. Conversely, in B. alvei Indole also appeared in the absence of exogenous the level of exogenous tryptophan has no influ- tryptophan. Figure 1 shows the indole-excretion ence on the specific activity of tryptophanase kinetics for various levels of tryptophan. When 606 HOCH AND DEMOSS J. BACTERIOL. tions (20 ,ug/ml) increased the absolute amount of indole formed but did not appreciably alter the kinetics of indole excretion. At lower concen- trations, anthranilate seemed to decrease the amount of indole formed. This observation lends support to a role for tryptophanase in early indole formation, since Burns (Ph.D. Thesis, Univ. of w 0 Illinois, Urbana) has shown that anthranilate is a competitive inhibitor of the E. coli trypto- 0 phanase. However, Gibson and Yanofsky (1960) showed that indole-3-glycerol phosphate (IGP) E synthetase is also inhibited by anthranilate. E Hence, the reaction is more probably a function 4 of a decreased IGP level.